Methods for detecting dihydroxyvitamin D metabolites by mass spectrometry

ABSTRACT

Provided are methods of detecting the presence or amount of a dihydroxyvitamin D metabolite in a sample using mass spectrometry. The methods generally comprise ionizing a dihydroxyvitamin D metabolite in a sample and detecting the amount of the ion to determine the presence or amount of the vitamin D metabolite in the sample. In certain preferred embodiments the methods include immunopurifying the dihydroxyvitamin D metabolites prior to mass spectrometry. Also provided are methods to detect the presence or amount of two or more dihydroxyvitamin D metabolites in a single assay.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application claims priority under 35 U.S.C. §120 to U.S.application Ser. No. 11/946,765, filed Nov. 28, 2007, incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to the detection of dihydroxyvitamin Dmetabolites. In a particular aspect, the invention relates to methodsfor detecting vitamin D metabolites by mass spectrometry.

BACKGROUND OF THE INVENTION

Vitamin D is an essential nutrient with important physiological roles inthe positive regulation of calcium (Ca²⁺) homeostasis. Vitamin D can bemade de novo in the skin by exposure to sunlight or it can be absorbedfrom the diet. There are two forms of vitamin D; vitamin D₂(ergocalciferol) and vitamin D₃ (cholecalciferol). Vitamin D₃ is theform synthesized de novo by animals. It is also a common supplementadded to milk products and certain food products produced in the UnitedStates. Both dietary and intrinsically synthesized vitamin D₃ mustundergo metabolic activation to generate bioactive metabolites. Inhumans, the initial step of vitamin D₃ activation occurs primarily inthe liver and involves hydroxylation to form the intermediate metabolite25-hydroxyvitamin D₃ (25-hydroxycholecalciferol; calcifediol; 25OHD₃).Calcifediol is the major form of vitamin D₃ in the circulation.Circulating 25OHD₃ is then converted by the kidney to1,25-dihydroxyvitamin D₃ (calcitriol; 1,25(OH)₂D₃), which is generallybelieved to be the metabolite of vitamin D₃ with the highest biologicalactivity.

Vitamin D₂ is derived from fungal and plant sources. Someover-the-counter dietary supplements contain ergocalciferol (vitamin D₂)rather than cholecalciferol (vitamin D₃). Drisdol, the only high-potencyprescription form of vitamin D available in the United States, isformulated with ergocalciferol. Vitamin D₂ undergoes a similar pathwayof metabolic activation in humans as vitamin D₃, forming the metabolites25-hydroxyvitamin D₂ (25OHD₂) and 1,25-dihydroxyvitamin D₃(1,25(OH)₂D₂). Vitamin D₂ and vitamin D₃ have long been assumed to bebiologically equivalent in humans, however recent reports suggest thatthere may be differences in the bioactivity and bioavailability of thesetwo forms of vitamin D (Armas et. al., (2004) J. Clin. Endocrinol.Metab. 89:5387-5391).

Measurement of vitamin D, the inactive vitamin D precursor, is rare inclinical settings and has little diagnostic value. Rather, serum levelsof 25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₂ (total25-hydroxyvitamin D; “25OHD”) are a useful index of vitamin Dnutritional status and the efficacy of certain vitamin D analogs.Therefore, the measurement of 25OHD is commonly used in the diagnosisand management of disorders of calcium metabolism. In this respect, lowlevels of 25OHD are indicative of vitamin D deficiency associated withdiseases such as hypocalcemia, hypophosphatemia, secondaryhyperparathyroidism, elevated alkaline phosphatase, osteomalacia inadults and rickets in children. In patients suspected of vitamin Dintoxication, elevated levels of 25OHD distinguishes this disorder fromother disorders that cause hypercalcemia.

Measurement of 1,25(OH)₂D is also used in clinical settings. For examplecertain disease states such as kidney failure can be diagnosed byreduced levels of circulating 1,25(OH)₂D and elevated levels of1,25(OH)₂D may be indicative of excess parathyroid hormone or may beindicative of certain diseases such as sarcoidosis or certain types oflymphoma.

Detection of vitamin D metabolites has been accomplished byradioimmunoassay with antibodies co-specific for 25-hydroxyvitamin D₃and 25-hydroxyvitamin D₂. Because the current immunologically-basedassays do not separately resolve 25-hydroxyvitamin D₃ and25-hydroxyvitamin D₂, the source of a deficiency in vitamin D nutritioncannot be determined without resorting to other tests. More recently,reports have been published that disclose methods for detecting specificvitamin D metabolites using mass spectrometry. For example Yeung B, etal., J Chromatogr. 1993, 645(1):115-23; Higashi T, et al., Steroids.2000, 65(5):281-94; Higashi T, et al., Biol Pharm Bull. 2001,24(7):738-43; and Higashi T, et al., J Pharm Biomed Anal. 2002,29(5):947-55 disclose methods for detecting various vitamin Dmetabolites using liquid chromatography and mass spectrometry. Thesemethods require that the metabolites be derivatized prior to detectionby mass-spectrometry. Methods to detect underivatized 1,25(OH)₂D₃ byliquid chromatography mass-spectrometry are disclosed in Kissmeyer andSonne, J Chromatogr A. 2001, 935(1-2):93-103.

SUMMARY OF THE INVENTION

The present invention provides methods for detecting the presence oramount of a dihydroxyvitamin D metabolite in a sample by massspectrometry, including tandem mass spectrometry.

In one aspect, methods are provided for determining the presence oramount of one or more dihydroxyvitamin D metabolites by tandem massspectrometry, that include: (a) immunopurifying one or moredihydroxyvitamin D metabolites from the sample; (b) further purifyingthe immunopurified dihydroxyvitamin D metabolite(s) by HPLC; (c)determining the amount of the vitamin D metabolites obtained from step(b) by tandem mass spectrometry by: (i) generating a precursor ion ofthe dihydroxyvitamin D metabolite(s); (ii) generating one or morefragment ions of the precursor ion; and (iii) detecting the presence oramount of one or more of the ions generated in step (c) or (d) or bothand relating the detected ions to the presence or amount of thedihydroxyvitamin D metabolite(s) in the sample. In certain preferredembodiments, dihydroxyvitamin D metabolites are immunopurified from thesample using anti-dihydroxyvitamin D antibodies attached to a solidsupport; preferably the dihydroxyvitamin D metabolites areimmunopurified using immunoparticles; preferably the immunoparticleshave anti-dihydroxyvitamin D antibodies on their surface. In certainembodiments, the dihydroxyvitamin D metabolite(s) include 1α,25(OH)₂D₂;in certain embodiments the dihydroxyvitamin D metabolite(s) include1α,25(OH)₂D₃; in some particularly preferred embodiments, provided aremethods for determining the presence or amount of 1α,25(OH)₂D₂ and1α,25(OH)₂D₃ in a single assay.

In certain preferred embodiments of the above aspect, thedihydroxyvitamin D metabolite(s) are derivatized prior to massspectrometry; in some particularly preferred embodiments thedihydroxyvitamin D metabolite(s) are derivatized with a Cookson-typereagent (e.g., a 4-substituted 1,2,4-triazoline-3,5-dione; TAD); incertain particularly preferred embodiments the dihydroxyvitamin Dmetabolite(s) are derivatized with 4-phenyl-1,2,4-triazoline-3,5-dione(PTAD); and in yet other particularly preferred embodiments thedihydroxyvitamin D metabolite(s) are derivatized with4′-carboxyphenyl-TAD. In certain preferred embodiments thedihydroxyvitamin D metabolite(s) include 1α,25(OH)₂D₂; the 1α,25(OH)₂D₂is derivatized with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) prior tomass spectrometry; and more preferably the precursor ion of 1α,25(OH)₂D₂has a mass/charge ratio of 586.37±0.5. In certain preferred embodimentsthe dihydroxyvitamin D metabolite(s) include 1α,25(OH)₂D₃; the1α,25(OH)₂D₃ is derivatized with 4-phenyl-1,2,4-triazoline-3,5-dione(PTAD) prior to mass spectrometry; and more preferably the precursor ionof 1α,25(OH)₂D₃ has a mass/charge ratio of 574.37±0.5. In certainpreferred embodiments, the dihydroxyvitamin D metabolite(s) arederivatized with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) prior tomass spectrometry and the fragment ions include at least one ion havinga mass/charge ratio of 314.12±0.5

In some preferred embodiments the dihydroxyvitamin D metabolite(s) arenot derivatized prior to mass spectrometry. In certain particularlypreferred embodiments the dihydroxyvitamin D metabolite(s) include1α,25(OH)₂D₂, the 1α,25(OH)₂D₂ is not derivatized prior to massspectrometry and more preferably the precursor ion of thenon-derivatized 1α,25(OH)₂D₂ has a mass/charge ratio of 411.35±0.5. Incertain particularly preferred embodiments the dihydroxyvitamin Dmetabolite(s) include 1α,25(OH)₂D₃, the 1α,25(OH)₂D₃ is not derivatizedprior to mass spectrometry and more preferably the precursor ion of thenon-derivatized 1α,25(OH)₂D₃ has a mass/charge ratio of 399.35±0.5. Incertain particularly preferred embodiments, the dihydroxyvitamin Dmetabolite(s) are not derivatized and the fragment ions include one ormore ions selected from the group consisting of ions having amass/charge ratio of 151.12±0.5 and 135.12±0.5.

In particularly preferred embodiments, methods are provided fordetermining the amount of 1α,25(OH)₂D₂ and 1α,25(OH)₂D₃ in a human bodysample by tandem mass spectrometry in a single assay that include: (a)immunopurifying the 1α,25(OH)₂D₂ and 1α,25(OH)₂D₃ from the sample; (b)derivatizing the 1α,25(OH)₂D₂ and 1α,25(OH)₂D₃ with4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); (c) purifying thederivatized 1α,25(OH)₂D₂ and 1α,25(OH)₂D₃ from step (b) by HPLC; (d)determining the amount of 1α,25(OH)₂D₂ and 1α,25(OH)₂D₃ obtained fromstep (c) by tandem mass spectrometry by; (i) generating a precursor ionof the derivatized 1α,25(OH)₂D₂ having a mass/charge ratio of 586.37±0.5and a precursor ion of the derivatized 1α,25(OH)₂D₃ having a mass/chargeratio of 574.37±0.5; (ii) generating one or more fragment ions of theprecursor ions from step (i) wherein at least one of the fragment ionshave a mass charge ration of 314.12±0.5; and (iii) detecting thepresence or amount of one or more of the ions generated in step (i) or(ii) or both and relating the detected ions to the presence or amount of1α,25(OH)₂D₂ and 1α,25(OH)₂D₃ in the sample.

As used herein, the term “dihydroxyvitamin D metabolite” refers to anydihydroxylated vitamin D species that may be found in the circulation ofan animal which is formed by a biosynthetic or metabolic pathway forvitamin D or a synthetic vitamin D analog. Preferably thedihydroxyvitamin D metabolite is hydroxylated at the 1 and 25 position.In particularly preferred embodiments, the vitamin D metabolite is1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃) or 1α,25-dihydroxyvitamin D₂(1α,25(OH)₂D₂). In certain preferred embodiments the dihydroxyvitamin Dmetabolites are naturally present in a body fluid of a mammal, morepreferably a human. In certain particularly preferred embodiments, themethods as described herein detect 1α,25-dihydroxyvitamin D₃(1α,25(OH)₂D₃) and/or 1α,25-dihydroxyvitamin (1α,25(OH)₂D₂) and do notdetect one or more dihydroxyvitamin-D metabolites selected from thegroup consisting of 24,25-dihydroxyvitamin D; 25,26-dihydroxyvitamin D;and 1α,3α-dihydroxyvitamin D.

As used herein, the term “purification” or “purify” refers to aprocedure that enriches the amount of one or more analytes of interestrelative to one or more other components of the sample. Purification, asused herein does not require the isolation of an analyte from allothers. In preferred embodiments, a purification step or procedure canbe used to remove one or more interfering substances, e.g. one or moresubstances that would interfere with the operation of the instrumentsused in the methods or substances that may interfere with the detectionof an analyte ion by mass spectrometry.

As used herein, the term “immunopurification” or “immunopurify” refersto a purification procedure that utilizes antibodies, includingpolyclonal or monoclonal antibodies, to enrich the one or more analytesof interest. Immunopurification can be performed using any of theimmunopurification methods well known in the art. Often theimmunopurification procedure utilizes antibodies bound, conjugated orotherwise attached to a solid support, for example a column, well, tube,gel, capsule, particle or the like. Immunopurification as used hereinincludes without limitation procedures often referred to in the art asimmunoprecipitation, as well as procedures often referred to in the artas affinity chromatography.

As used herein, the term “immunoparticle” refers to a capsule, bead, gelparticle or the like that has antibodies bound, conjugated or otherwiseattached to its surface (either on and/or in the particle). In certainpreferred embodiments, immunoparticles are sepharose or agarose beads.In alternative preferred embodiments, immunoparticles are glass, plasticor silica beads, or silica gel.

As used herein, the term “anti-dihydroxyvitamin D antibody” refers toany polyclonal or monoclonal antibody that has an affinity for one ormore dihydroxyvitamin D metabolites. In certain preferred embodimentsthe anti-dihydroxyvitamin D antibodies bind 1α,25(OH)₂D₃ and1α,25(OH)₂D₂. In some preferred embodiments the anti-dihydroxyvitamin Dantibodies bind 1α,25(OH)₂D₃ and 1α,25(OH)₂D₂ with equal or similaraffinity. In other preferred embodiments the anti-dihydroxyvitamin Dantibodies bind 1α,25(OH)₂D₃ with significantly higher affinity than1α,25(OH)₂D₂; in alternative preferred embodiments theanti-dihydroxyvitamin D antibodies bind 1α,25(OH)₂D₂ with significantlyhigher affinity than 1α,25(OH)₂D₃. In various embodiments thespecificity of anti-dihydroxyvitamin D antibodies to chemical speciesother than dihydroxyvitamin D metabolites may vary; for example incertain preferred embodiments the anti-dihydroxyvitamin D antibodies arespecific for dihydroxyvitamin D metabolites and thus have little or noaffinity for chemical species other than dihydroxyvitamin D metabolites(e.g., other vitamin D metabolites such as vitamin D or25-hydroxyvitamin D), whereas in other preferred embodiments theanti-dihydroxyvitamin D antibodies are non-specific and thus bindcertain chemical species other than dihydroxyvitamin D metabolites (forexample a non-specific anti-dihydroxyvitamin D antibody may bind othervitamin D metabolites such as vitamin D or 25-hydroxyvitamin D).

In some preferred embodiments of the methods disclosed herein, thedihydroxyvitamin D metabolite(s) are not derivatized prior to massspectrometry. In other preferred embodiments, the vitamin D metabolitesare derivatized prior to mass spectrometry.

As used herein, “biological sample” refers to any sample from abiological source. As used herein, “body fluid” means any fluid that canbe isolated from the body of an individual. For example, “body fluid”may include blood, plasma, serum, bile, saliva, urine, tears,perspiration, and the like.

As used herein, “derivatizing” means reacting two molecules to form anew molecule. Derivatizing agents may include Cookson-type reagents(e.g., 4-substituted 1,2,4-triazoline-3,5-diones; TAD); isothiocyanategroups, dinitro-fluorophenyl groups, nitrophenoxycarbonyl groups, and/orphthalaldehyde groups. In certain preferred embodiments, derivitizationis performed using methods such as those disclosed in, for example,Vreeken, et. al., Biol. Mass Spec. 22:621-632; Yeung B, et al., JChromatogr. 1993, 645(1):115-23; Higashi T, et al., Biol Pharm Bull.2001, 24(7):738-43; or Higashi T, et al., J Pharm Biomed Anal. 2002,29(5):947-55. In preferred embodiments the derivatizing agents areCookson-type reagents. Particularly preferred derivatizing reagentsinclude 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD);4′-carboxyphenyl-TAD;4-[4-(6-methoxy-2-benzoxazolyl)phenyl]-1,2,4-triazoline-3,5-dione(MBOTAD).;4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-ihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione(DMEQTAD); 4-nitrophenyl-TAD; 4-pentafluorophenyl-TAD;4-ferrocenylethyl-TAD; 4-quarternaryamine-TAD; and the like. In certainpreferred embodiments derivitization is performed prior tochromatography; however in other preferred embodiments derivitization isperformed after chromatography, for example using methods similar tothose described in Vreeken, et. al., Biol. Mass Spec. 22:621-632.

As used herein, “chromatography” refers to a process in which a chemicalmixture carried by a liquid or gas is separated into components as aresult of differential distribution of the chemical entities as theyflow around or over a stationary liquid or solid phase.

As used herein, “liquid chromatography” (LC) means a process ofselective retardation of one or more components of a fluid solution asthe fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). “Liquid chromatography”includes reverse phase liquid chromatography (RPLC), high performanceliquid chromatography (HPLC) and high turbulence liquid chromatography(HTLC).

As used herein, the term “HPLC” or “high performance liquidchromatography” refers to liquid chromatography in which the degree ofseparation is increased by forcing the mobile phase under pressurethrough a stationary phase, typically a densely packed column.

As used herein, the term “gas chromatography” refers to chromatographyin which the sample mixture is vaporized and injected into a stream ofcarrier gas (as nitrogen or helium) moving through a column containing astationary phase composed of a liquid or a particulate solid and isseparated into its component compounds according to the affinity of thecompounds for the stationary phase

As used herein, “mass spectrometry” (MS) refers to an analyticaltechnique to identify compounds by their mass. MS technology generallyincludes (1) ionizing the compounds to form charged compounds; and (2)detecting the molecular weight of the charged compound and calculating amass-to-charge ratio (m/z). The compound may be ionized and detected byany suitable means. A “mass spectrometer” generally includes an ionizerand an ion detector. See, e.g., U.S. Pat. No. 6,204,500, entitled “MassSpectrometry From Surfaces;” U.S. Pat. No. 6,107,623, entitled “Methodsand Apparatus for Tandem Mass Spectrometry;” U.S. Pat. No. 6,268,144,entitled “DNA Diagnostics Based On Mass Spectrometry;” U.S. Pat. No.6,124,137, entitled “Surface-Enhanced Photolabile Attachment And ReleaseFor Desorption And Detection Of Analytes;” Wright et al., ProstateCancer and Prostatic Diseases 2:264-76 (1999); and Merchant andWeinberger, Electrophoresis 21:1164-67 (2000).

The term “electron ionization” as used herein refers to methods in whichan analyte of interest in a gaseous or vapor phase interacts with a flowof electrons. Impact of the electrons with the analyte produces analyteions, which may then be subjected to a mass spectrometry technique.

The term “chemical ionization” as used herein refers to methods in whicha reagent gas (e.g. ammonia) is subjected to electron impact, andanalyte ions are formed by the interaction of reagent gas ions andanalyte molecules.

The term “fast atom bombardment” as used herein refers to methods inwhich a beam of high energy atoms (often Xe or Ar) impacts anon-volatile sample, desorbing and ionizing molecules contained in thesample. Test samples are dissolved in a viscous liquid matrix such asglycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether,2-nitrophenyloctyl ether, sulfolane, diethanolamine, andtriethanolamine.

The term “field desorption” as used herein refers to methods in which anon-volatile test sample is placed on an ionization surface, and anintense electric field is used to generate analyte ions.

The term “ionization” as used herein refers to the process of generatingan analyte ion having a net electrical charge equal to one or moreelectron units. Negative ions are those having a net negative charge ofone or more electron units, while positive ions are those having a netpositive charge of one or more electron units.

The term “operating in negative ion mode” refers to those massspectrometry methods where negative ions are detected. Similarly,“operating in positive ion mode” refers to those mass spectrometrymethods where positive ions are detected.

The term “desorption” as used herein refers to the removal of an analytefrom a surface and/or the entry of an analyte into a gaseous phase.

In a second aspect, methods are provided for determining the presence oramount of 1α,25(OH)₂D₂ in a sample by tandem mass spectrometry thatinclude (a) derivatizing the 1α,25(OH)₂D₂ in the sample with4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); (b) purifying thederivatized 1α,25(OH)₂D₂ by HPLC; (c) generating a precursor ion of thederivatized 1α,25(OH)₂D₂ having a mass/charge ratio of 586.37±0.5; (d)generating one or more fragment ions of the precursor ion, wherein atleast one of the fragment ions comprise an ion having a mass/chargeratio of 314.12±0.5; and (e) detecting the presence or amount of one ormore of the ions generated in step (c) or (d) or both and relating thedetected ions to the presence or amount of 1α,25(OH)₂D₂ in the sample.In certain preferred embodiments the 1α,25(OH)₂D₂ in the sample ispurified by immunopurification prior to step (a); preferably theimmunopurification includes immunopurification with immunoparticles;preferably the immunoparticles have an anti-dihydroxyvitamin Dmetabolite antibody bound to the surface. In some preferred embodimentsof this aspect, the method further includes determining the presence oramount of 1α,25(OH)₂D₃ in the sample; preferably the 1α,25(OH)₂D₃ isderivatized with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) prior tomass spectrometry; more preferably the precursor ion of the 1α,25(OH)₂D₃has a mass/charge ratio of 574.37±0.5

In a third aspect, methods are provided for determining the presence oramount of 1α,25(OH)₂D₃ in a sample by tandem mass spectrometry thatinclude (a) derivatizing the 1α,25(OH)₂D₃ in the sample with4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); (b) purifying thederivatized 1α,25(OH)₂D₃ by HPLC; (c) generating a precursor ion of thederivatized 1α,25(OH)₂D₃ having a mass/charge ratio of 574.37±0.5; (d)generating one or more fragment ions of the precursor ion, wherein atleast one of the fragment ions comprise an ion having a mass/chargeratio of 314.12±0.5; and (e) detecting the presence or amount of one ormore of the ions generated in step (c) or (d) or both and relating thedetected ions to the presence or amount of 1α,25(OH)₂D₃ in the sample.In certain preferred embodiments the 1α,25(OH)₂D₃ in the sample ispurified by immunopurification prior to step (a); preferably theimmunopurification includes immunopurification with immunoparticles;preferably the immunoparticles have an anti-dihydroxyvitamin Dmetabolite antibody bound to the surface. In some preferred embodimentsof this aspect, the method further includes determining the presence oramount of 1α,25(OH)₂D₂ in the sample; preferably the 1α,25(OH)₂D₂ isderivatized with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) prior tomass spectrometry; more preferably the precursor ion of the 1α,25(OH)₂D₂has a mass/charge ratio of 586.37±0.5.

In a fourth aspect, methods are provided for determining the presence oramount of 1α,25(OH)₂D₂ in a sample by tandem mass spectrometry thatinclude (a) purifying the 1α,25(OH)₂D₂ by HPLC; (b) generating aprecursor ion of the 1α,25(OH)₂D₂ having a mass/charge ratio of411.35±0.5; (c) generating one or more fragment ions of the precursorion, wherein the fragment ions include one or more ions selected fromthe group consisting of ions having a mass/charge ratio of 151.12±0.5and 135.12±0.5; and (d) detecting the presence or amount of one or moreof the ions generated in step (b) or (c) or both and relating thedetected ions to the presence or amount of the 1α,25(OH)₂D₂ in thesample. In preferred embodiments of this aspect the 1α,25(OH)₂D₂ is notderivatized prior to mass spectrometry. In certain preferred embodimentsthe 1α,25(OH)₂D₂ in the sample is purified by immunopurification priorto step (a); preferably the immunopurification includesimmunopurification with immunoparticles; preferably the immunoparticleshave an anti-dihydroxyvitamin D metabolite antibody bound to thesurface. In some preferred embodiments of this aspect, the methodfurther includes determining the presence or amount of 1α,25(OH)₂D₃ inthe sample; preferably the 1α,25(OH)₂D₃ is not derivatized prior to massspectrometry; more preferably the precursor ion of the 1α,25(OH)₂D₃ hasa mass/charge ratio of 399.35±0.5.

In a fifth aspect, methods are provided for determining the presence oramount of 1α,25(OH)₂D₃ in a sample by tandem mass spectrometry thatinclude (a) purifying the 1α,25(OH)₂D₃ by HPLC; (b) generating aprecursor ion of the 1α,25(OH)₂D₃ having a mass/charge ratio of399.35±0.5; (c) generating one or more fragment ions of the precursorion, wherein the fragment ions include one or more ions selected fromthe group consisting of ions having a mass/charge ratio of 151.12±0.5and 135.12±0.5; and (d) detecting the presence or amount of one or moreof the ions generated in step (b) or (c) or both and relating thedetected ions to the presence or amount of the 1α,25(OH)₂D₃ in thesample. In preferred embodiments of this aspect the 1α,25(OH)₂D₃ is notderivatized prior to mass spectrometry. In certain preferred embodimentsthe 1α,25(OH)₂D₃ in the sample is purified by immunopurification priorto step (a); preferably the immunopurification includesimmunopurification with immunoparticles; preferably the immunoparticleshave an anti-dihydroxyvitamin D metabolite antibody bound to thesurface. In some preferred embodiments of this aspect, the methodfurther includes determining the presence or amount of 1α,25(OH)₂D₂ inthe sample; preferably the 1α,25(OH)₂D₂ is not derivatized prior to massspectrometry; more preferably the precursor ion of the 1α,25(OH)₂D₂ hasa mass/charge ratio of 411.35±0.5.

The term “about” as used herein in reference to quantitativemeasurements, refers to the indicated value plus or minus 10%.

DETAILED DESCRIPTION OF THE INVENTION

Methods are described for detecting and quantifying dihydroxyvitamin Dmetabolites in a test sample. Some preferred methods disclosed hereinutilize liquid chromatography (LC), most preferably HPLC, to purifyselected analytes, and combine this purification with unique methods ofmass spectrometry (MS), thereby providing a high-throughput assay systemfor detecting and quantifying dihydroxyvitamin D metabolites in a testsample. In certain particularly preferred embodiments, dihydroxyvitaminD metabolites are immunopurified prior to mass spectrometry. Thepreferred embodiments are particularly well suited for application inlarge clinical laboratories. Methods of detecting and quantifyingdihydroxyvitamin D metabolites are provided that have enhancedspecificity and are accomplished in less time and with less samplepreparation than required in other dihydroxyvitamin D metabolite assays.

Suitable test samples include any test sample that may contain theanalyte of interest. For example, samples obtained during themanufacture of an analyte can be analyzed to determine the compositionand yield of the manufacturing process. In some preferred embodiments, asample is a biological sample; that is, a sample obtained from anybiological source, such as an animal, a cell culture, an organ culture,etc. In certain preferred embodiments, samples are obtained from amammalian animal, such as a dog, cat, horse, etc. Particularly preferredmammalian animals are primates, most preferably humans. Particularlypreferred samples include blood, plasma, serum, hair, muscle, urine,saliva, tear, cerebrospinal fluid, or other tissue sample. Such samplesmay be obtained, for example, from a patient; that is, a living personpresenting oneself in a clinical setting for diagnosis, prognosis, ortreatment of a disease or condition. The test sample is preferablyobtained from a patient, for example, blood serum.

Sample Preparation for Mass Spectrometry

Methods may be used prior to mass spectrometry to enrichdihydroxyvitamin D metabolites relative to other components in thesample, or to increase the concentration of the dihydroxyvitamin Dmetabolites in the sample. Such methods include, for example,filtration, centrifugation, thin layer chromatography (TLC),electrophoresis including capillary electrophoresis, affinityseparations including immunoaffinity separations, extraction methodsincluding ethyl acetate extraction and methanol extraction, and the useof chaotropic agents or any combination of the above or the like.

Samples may be processed or purified to obtain preparations that aresuitable for analysis by mass spectrometry. Such purification willusually include chromatography, such as liquid chromatography, and mayalso often involve an additional purification procedure that isperformed prior to chromatography. Various procedures may be used forthis purpose depending on the type of sample or the type ofchromatography. Examples include filtration, extraction, precipitation,centrifugation, delipidization, dilution, combinations thereof and thelike. Protein precipitation is one preferred method of preparing aliquid biological sample, such as serum or plasma, for chromatography.Such protein purification methods are well known in the art, forexample. Polson et al., Journal of Chromatography B 785:263-275 (2003),describes protein precipitation methods suitable for use in the methodsof the invention. Protein precipitation may be used to remove most ofthe protein from the sample leaving dihydroxyvitamin D metabolitessoluble in the supernatant. The samples can be centrifuged to separatethe liquid supernatant from the precipitated proteins. The resultantsupernatant can then be applied to liquid chromatography and subsequentmass spectrometry analysis. In one embodiment of the invention, theprotein precipitation involves adding one volume of the liquid sample(e.g. plasma) to about four volumes of methanol. In certain embodiments,the use of protein precipitation obviates the need for high turbulenceliquid chromatography (“HTLC”) or on-line extraction prior to HPLC andmass spectrometry. Accordingly in such embodiments, the method involves(1) performing a protein precipitation of the sample of interest; and(2) loading the supernatant directly onto the HPLC-mass spectrometerwithout using on-line extraction or high turbulence liquidchromatography (“HTLC”).

Immunopurification.

In particularly preferred embodiments, the methods includeimmunopurifying dihydroxyvitamin D metabolites prior to massspectrometry analysis. The immunopurification step may be performedusing any of the immunopurification methods well known in the art. Oftenthe immunopurification procedure utilizes antibodies bound, conjugated,immobilized or otherwise attached to a solid support, for example acolumn, well, tube, capsule, particle or the like. Generally,immunopurification methods involve (1) incubating a sample containingthe analyte of interest with antibodies such that the analyte binds tothe antibodies, (2) performing one or more washing steps, and (3)eluting the analyte from the antibodies.

In certain embodiments the incubation step of the immunopurification isperformed with the antibodies free in solution and the antibodies aresubsequently bound or attached to a solid surface prior to the washingsteps. In certain embodiments this can be achieved using a primaryantibody that is an anti-dihydroxyvitamin D antibody and a secondaryantibody attached to a solid surface that has an affinity to the primaryanti-dihydroxyvitamin D antibody. In alternative embodiments, theprimary antibody is bound to the solid surface prior to the incubationstep.

Appropriate solid supports include without limitation tubes, slides,columns, beads, capsules, particles, gels, and the like. In somepreferred embodiments, the solid support is a multi-well plate, such as,for example, a 96 well plate, a 384-well plate or the like. In certainpreferred embodiments the solid support are sephararose or agarose beadsor gels. There are numerous methods well known in the art by whichantibodies (for example, an anti-dihydroxyvitamin D antibody or asecondary antibody) may be bound, attached, immobilized or coupled to asolid support, e.g., covalent or non-covalent linkages adsorption,affinity binding, ionic linkages and the like. In certain preferredembodiments antibodies are coupled using CNBr, for example theantibodies may be coupled to CNBr activated sepharose. In otherembodiments, the antibody is attached to the solid support through anantibody binding protein such as protein A, protein G, protein A/G, orprotein L.

The washing step of the immunopurification methods generally involvewashing the solid support such that the dihydroxyvitamin D metabolitesremain bound to the anti-dihydroxyvitamin D antibodies on the solidsupport. The elution step of the immunopurification generally involvesthe addition of a solution that disrupts the binding of dihydroxyvitaminD metabolites to the anti-dihydroxyvitamin D antibodies. Exemplaryelution solutions include organic solutions (preferably ethanol), saltsolutions, and high or low pH solutions.

In certain preferred embodiments, immunopurification is performed usingimmunoparticles having anti-dihydroxyvitamin D antibodies. In certainpreferred embodiments the test sample possibly containingdihydroxyvitamin D metabolites and the immunoparticles are mixed in atube for incubation and binding of dihydroxyvitamin D metabolites to theanti-dihydroxyvitamin D antibodies attached to the immunoparticles; thetube is centrifuged leaving the immunoparticles in a pellet; thesupernatant is removed; the immunoparticles are washed one or more timesby adding a solution to the pellet and recentrifuging; and thedihydroxyvitamin D metabolites are eluted by adding an elution solutionto the immunoparticles, the tube is centrifuged leaving theimmunoparticles in a pellet; and the supernatant containingdihydroxyvitamin D metabolites is collected. In related preferredembodiments, the immunopurification is performed using a column orcartridge that contains immunoparticles having anti-dihydroxyvitamin Dantibodies. Preferably, the such column or cartridge is configured andarranged in a manner to allow solutions to flow through while keepingthe immunoparticles contained therein. In certain preferred embodiments,the solution is forced through the column or cartridge by gravity,centrifugation or pressure. The use of columns may improve the ease ofperforming the incubation, washing and elution steps. In some preferredembodiments, the immunopurification is performed by affinitychromatography; preferably automated affinity chromatography; preferablyaffinity-HPLC; or preferably affinity chromatography using an automatedsystem such as the AKTA FPLC Chromatographic system sold commercially byGE Healthcare (formerly Amersham biosciences).

In certain embodiments, the sample preparation and immunopurificationcan be performed using methods and reagents from commercially availablekits. For example, IDS Inc (Fountain Hills, Ariz.) offers a1,25-Dihydroxy Vitamin D ¹²⁵I Radioimmunoassay kit (Catalogue NumberAA-54F1) that includes directions and reagents for extracting andimmunoextracting dihydroxyvitamin D prior to the radioimmunoassay (RIA).See the “Product Support” document for the Catalogue Number AA-54F1 IDS,Inc., kit which is hereby incorporated by reference in its entirety. Inparticular, the IDS dihydroxyvitamin D RIA kit includes a dextransulphate/magnesium chloride delipidization step and an immunoextractionstep using an immunocapsule device containing a suspension of particlesto which is attached a monoclonal antibody specific for 1,25dihydroxyvitamin D. Accordingly, in certain embodiments of the methodsdescribed herein, the samples are subject to vitamin Dimmunopurification using the IDS kit or methods, reagents anddihydroxyvitamin D immunopurification devices similar to those providedin the IDS kit. Antibodies and dihydroxy purification immunopurificationdevices are also provided with the 1,25-(OH)2-Vitamin D ImmunoTube ELISAKit (Catalog Number 30-2113) offered commercially by ALPO Diagnostics(Salem, N.H.). The kit includes an anti 1,25-(OH)₂ vitamin-D detectionantibody (Catalog number K2113A1), ImmunoTube columns forimmunopurification of 1,25-dihydroxyvitamin D (Catalog Number K2113.SI)as well as buffers and other reagents that may be used to immunopurify1,25-dihydroxyvitamin D. In certain embodiments of the methods describedherein, one or more of the components of the ALPO Diagnostics kit areused in to immunopurify 1,25-dihydroxyvitamin D.

Liquid Chromatography.

Generally, chromatography is performed prior to mass spectrometry,preferably the chromatography is liquid chromatography, more preferablyhigh performance liquid chromatography (HPLC). In some preferredembodiments the chromatography is not gas chromatography. Preferably,the methods of the invention are performed without subjecting thesamples, or the dihydroxyvitamin D metabolites of interest, to gaschromatography prior to mass spectrometric analysis.

Liquid chromatography (LC) including high-performance liquidchromatography (HPLC) rely on relatively slow, laminar flow technology.Traditional HPLC analysis relies on column packings in which laminarflow of the sample through the column is the basis for separation of theanalyte of interest from the sample. The skilled artisan will understandthat separation in such columns is a diffusional process. HPLC has beensuccessfully applied to the separation of compounds in biologicalsamples. But a significant amount of sample preparation is requiredprior to the separation and subsequent analysis with a mass spectrometer(MS), making this technique labor intensive. In addition, most HPLCsystems do not utilize the mass spectrometer to its fullest potential,allowing only one HPLC system to be connected to a single MS instrument,resulting in lengthy time requirements for performing a large number ofassays.

Various methods have been described involving the use of HPLC for sampleclean-up prior to mass spectrometry analysis. See, e.g., Taylor et al.,Therapeutic Drug Monitoring 22:608-12 (2000) (manual precipitation ofblood samples, followed by manual C18 solid phase extraction, injectioninto an HPLC for chromatography on a C18 analytical column, and MS/MSanalysis); and Salm et al, Clin. Therapeutics 22 Supl. B:B71-B85 (2000)(manual precipitation of blood samples, followed by manual C18 solidphase extraction, injection into an HPLC for chromatography on a C18analytical column, and MS/MS analysis).

One of skill in the art can select HPLC instruments and columns that aresuitable for use in the invention. The chromatographic column typicallyincludes a medium (i.e., a packing material) to facilitate separation ofchemical moieties (i.e., fractionation). The medium may include minuteparticles. The particles include a bonded surface that interacts withthe various chemical moieties to facilitate separation of the chemicalmoieties. One suitable bonded surface is a hydrophobic bonded surfacesuch as an alkyl bonded surface. Alkyl bonded surfaces may include C-4,C-8, or C-18 bonded alkyl groups, preferably C-18 bonded groups. Thechromatographic column includes an inlet port for receiving a sample andan outlet port for discharging an effluent that includes thefractionated sample. In one embodiment, the sample (or pre-purifiedsample) is applied to the column at the inlet port, eluted with asolvent or solvent mixture, and discharged at the outlet port. Differentsolvent modes may be selected for eluting the analytes of interest. Forexample, liquid chromatography may be performed using a gradient mode,an isocratic mode, or a polytyptic (i.e. mixed) mode. Duringchromatography, the separation of materials is effected by variablessuch as choice of eluent (also known as a “mobile phase”), choice ofgradient elution and the gradient conditions, temperature, etc.

In certain embodiments, an analyte may be purified by applying a sampleto a column under conditions where the analyte of interest is reversiblyretained by the column packing material, while one or more othermaterials are not retained. In these embodiments, a first mobile phasecondition can be employed where the analyte of interest is retained bythe column, and a second mobile phase condition can subsequently beemployed to remove retained material from the column, once thenon-retained materials are washed through. Alternatively, an analyte maybe purified by applying a sample to a column under mobile phaseconditions where the analyte of interest elutes at a differential ratein comparison to one or more other materials. Such procedures may enrichthe amount of one or more analytes of interest relative to one or moreother components of the sample.

Recently, high turbulence liquid chromatography (“HTLC”), also calledhigh throughput liquid chromatography, has been applied for samplepreparation prior to analysis by mass spectrometry. See, e.g., Zimmer etal., J. Chromatogr. A 854:23-35 (1999); see also, U.S. Pat. Nos.5,968,367; 5,919,368; 5,795,469; and 5,772,874. Traditional HPLCanalysis relies on column packings in which laminar flow of the samplethrough the column is the basis for separation of the analyte ofinterest from the sample. The skilled artisan will understand thatseparation in such columns is a diffusional process. In contrast, it isbelieved that turbulent flow, such as that provided by HTLC columns andmethods, may enhance the rate of mass transfer, improving the separationcharacteristics provided. In some embodiments, high turbulence liquidchromatography (HTLC), alone or in combination with one or morepurification methods, may be used to purify the dihydroxyvitamin Dmetabolite of interest prior to mass spectrometry. In such embodimentssamples may be extracted using an HTLC extraction cartridge whichcaptures the analyte, then eluted and chromatographed on a second HTLCcolumn or onto an analytical HPLC column prior to ionization. Becausethe steps involved in these chromatography procedures can be linked inan automated fashion, the requirement for operator involvement duringthe purification of the analyte can be minimized. In certain embodimentsof the method, samples are subjected to protein precipitation asdescribed above prior to loading on the HTLC column; in alternativeembodiments, the samples may be loaded directly onto the HTLC withoutbeing subjected to protein precipitation.

Recently, research has shown that epimerization of the hydroxyl group ofthe A-ring of vitamin D₃ metabolites is an important aspect of vitaminD₃ metabolism and bioactivation, and that depending on the cell typesinvolved, 3-C epimers of vitamin D₃ metabolites (e.g., 3-epi-25(OH)D₃;3-epi-24,25(OH)₂D₃; and 3-epi-1,25(OH)₂D₃) are often major metabolicproducts. See Kamao et al., J. Biol. Chem., 279:15897-15907 (2004).Kamao et al., further provides methods of separating various vitamin Dmetabolites, including 3-C epimers, using Chiral HPLC. Accordingly, theinvention also provides methods of detecting the presence, absenceand/or amount of a specific epimer of one or more vitamin D metabolites,preferably vitamin D₃ metabolites, in a sample by (1) separating one ormore specific vitamin D metabolites by chiral chromatography, preferablychiral HPLC; and (2) detecting the presence and/or amount of one or morevitamin D metabolites using mass spectrometry methods as describedherein. The chiral chromatography procedures described in Kamao et al.,are suitable for the methods of the invention, however, one of ordinaryskill in the art understands that there are numerous other chiralchromatography methods that would also be suitable. In preferredembodiments the method includes, separating 25(OH)D₃ from3-epi-25(OH)D₃, if present in a sample, using chiral chromatography; anddetecting the presence and/or amount of the 25(OH)D₃ and the3-epi-25(OH)D₃ in the sample using mass spectrometry. In relatedembodiments, the method includes separating 1α,25(OH)₂D₃ from3-epi-1α,25(OH)₂D₃, if present in a sample, using chiral chromatography;and detecting the presence and/or amount of the 1α,25(OH)₂D₃ and the3-epi-1α,25(OH)₂D₃ in the sample using mass spectrometry. In certainembodiments of the invention, chiral chromatography is used inconjunction with the HTLC methods described above.

Detection and Quantitation by Mass Spectrometry

Disclosed are methods for detecting the presence or amount of one ormore dihydroxyvitamin D metabolites in a sample. In certain aspects themethod involves ionizing the dihydroxyvitamin D metabolite(s), detectingthe ion(s) by mass spectrometry, and relating the presence or amount ofthe ion(s) to the presence or amount of the dihydroxyvitamin Dmetabolite(s) in the sample. The method may include (a) purifying adihydroxyvitamin D metabolite, if present in the sample, (b) ionizingthe purified dihydroxyvitamin D metabolite and (c) detecting thepresence or amount of the ion, wherein the presence or amount of the ionis related to the presence or amount of the dihydroxyvitamin Dmetabolite in the sample. In preferred embodiments, the ionizing step(b) may include (i) ionizing a dihydroxyvitamin D metabolite, if presentin the sample, to produce an ion; (ii) isolating the dihydroxyvitamin Dmetabolite ion by mass spectrometry to provide a precursor ion; and(iii) effecting a collision between the isolated precursor ion and aninert collision gas to produce at least one fragment ion detectable in amass spectrometer. In certain preferred embodiments the precursor ion isa protonated and dehydrated ion of the dihydroxyvitamin D metabolite.

Further provided is a method for determining the presence or amount of adihydroxyvitamin D metabolite in a test sample by tandem massspectrometry. The method may involve (a) generating a protonated anddehydrated precursor ion of the dihydroxyvitamin D metabolite; (b)generating one or more fragment ions of the precursor ion; and (c)detecting the presence or amount of one or more of the ions generated instep (a) or (b) or both and relating the detected ions to the presenceor amount of the dihydroxyvitamin D metabolite in the sample.

In certain preferred embodiments of the invention, at least one fragmention is detected, wherein the presence or amount of the precursor and/orat least one fragment ion is related to the presence or amount of thedihydroxyvitamin D metabolite in the sample. Preferably at least onefragment ion is specific for the dihydroxyvitamin D metabolite ofinterest. In some embodiments, the methods of the invention can be usedto detect and quantify two or more dihydroxyvitamin D metabolites in asingle assay.

Mass spectrometry is performed using a mass spectrometer which includesan ion source for ionizing the fractionated sample and creating chargedmolecules for further analysis. For example ionization of the sample maybe performed by electrospray ionization (ESI), atmospheric pressurechemical ionization (APCI), photoionization, electron ionization, fastatom bombardment (FAB)/liquid secondary ionization (LSIMS), matrixassisted laser desorption ionization (MALDI), field ionization, fielddesorption, thermospray/plasmaspray ionization, and particle beamionization. The skilled artisan will understand that the choice ofionization method can be determined based on the analyte to be measured,type of sample, the type of detector, the choice of positive versusnegative mode, etc.

After the sample has been ionized, the positively charged or negativelycharged ions thereby created may be analyzed to determine amass-to-charge ratio (i.e., m/z). Suitable analyzers for determiningmass-to-charge ratios include quadropole analyzers, ion traps analyzers,and time-of-flight analyzers. The ions may be detected using severaldetection modes. For example, selected ions may be detected (i.e., usinga selective ion monitoring mode (SIM)), or alternatively, ions may bedetected using a scanning mode, e.g., multiple reaction monitoring (MRM)or selected reaction monitoring (SRM). Preferably, the mass-to-chargeratio is determined using a quadropole analyzer. For example, in a“quadrupole” or “quadrupole ion trap” instrument, ions in an oscillatingradio frequency field experience a force proportional to the DCpotential applied between electrodes, the amplitude of the RF signal,and m/z. The voltage and amplitude can be selected so that only ionshaving a particular m/z travel the length of the quadrupole, while allother ions are deflected. Thus, quadrupole instruments can act as both a“mass filter” and as a “mass detector” for the ions injected into theinstrument.

One may enhance the resolution of the MS technique by employing “tandemmass spectrometry,” or “MS/MS.” In this technique, a precursor ion (alsocalled a parent ion) generated from a molecule of interest can befiltered in an MS instrument, and the precursor on is subsequentlyfragmented to yield one or more fragment ions (also called daughter ionsor product ions) that are then analyzed in a second MS procedure. Bycareful selection of precursor ions, only ions produced by certainanalytes are passed to the fragmentation chamber, where collision withatoms of an inert gas to produce the daughter ions. Because both theprecursor and fragment ions are produced in a reproducible fashion undera given set of ionization/fragmentation conditions, the MS/MS techniquecan provide an extremely powerful analytical tool. For example, thecombination of filtration/fragmentation can be used to eliminateinterfering substances, and can be particularly useful in complexsamples, such as biological samples.

Additionally, recent advances in technology, such as matrix-assistedlaser desorption ionization coupled with time-of-flight analyzers(“MALDI-TOF”) permit the analysis of analytes at femtomole levels invery short ion pulses. Mass spectrometers that combine time-of-flightanalyzers with tandem MS are also well known to the artisan.Additionally, multiple mass spectrometry steps can be combined inmethods known as “MS/MS^(n).” Various other combinations may beemployed, such as MS/MS/TOF, MALDI/MS/MS/TOF, or SELDI/MS/MS/TOF massspectrometry.

The mass spectrometer typically provides the user with an ion scan; thatis, the relative abundance of each ion with a particular m/z over agiven range (e.g., 100 to 1000 amu). The results of an analyte assay,that is, a mass spectrum, can be related to the amount of the analyte inthe original sample by numerous methods known in the art. For example,given that sampling and analysis parameters are carefully controlled,the relative abundance of a given ion can be compared to a table thatconverts that relative abundance to an absolute amount of the originalmolecule. Alternatively, molecular standards can be run with thesamples, and a standard curve constructed based on ions generated fromthose standards. Using such a standard curve, the relative abundance ofa given ion can be converted into an absolute amount of the originalmolecule. In certain preferred embodiments, an internal standard is usedto generate a standard curve for calculating the quantity of thedihydroxyvitamin D metabolite. Methods of generating and using suchstandard curves are well known in the art and one of ordinary skill iscapable of selecting an appropriate internal standard. For example, anisotope of a dihydroxyvitamin D metabolite may be used as an internalstandard, in preferred embodiments the dihydroxyvitamin D metabolite isa deuterated dihydroxyvitamin D metabolite, for example1α,25(OH)₂D₂-[26,26,26,27,27,27]-²H or 1α,25(OH)₂D₃-[6,19,19′]-²H orboth. Numerous other methods for relating the presence or amount of anion to the presence or amount of the original molecule will be wellknown to those of ordinary skill in the art.

One or more steps of the methods of the invention can be performed usingautomated machines. In certain embodiments, one or more purificationsteps are performed on line, and more preferably all of the purificationand mass spectrometry steps may be performed in an on-line fashion.

In certain embodiments, such as MS/MS, where precursor ions are isolatedfor further fragmentation, collision activation dissociation is oftenused to generate the fragment ions for further detection. In CAD,precursor ions gain energy through collisions with an inert gas, andsubsequently fragment by a process referred to as “unimoleculardecomposition”. Sufficient energy must be deposited in the precursor ionso that certain bonds within the ion can be broken due to increasedvibrational energy.

In particularly preferred embodiments dihydroxyvitamin D metabolites aredetected and/or quantified using LC-MS/MS as follows. The samples aresubjected to liquid chromatography, preferably HPLC, the flow of liquidsolvent from the chromatographic column enters the heated nebulizerinterface of a LC-MS/MS analyzer and the solvent/analyte mixture isconverted to vapor in the heated tubing of the interface. The analytes(i.e. dihydroxyvitamin D metabolites), contained in the nebulizedsolvent, are ionized by the corona discharge needle of the interface,which applies a large voltage to the nebulized solvent/analyte mixture.The ions, i.e. precursor ions, pass through the orifice of theinstrument and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 andQ3) are mass filters, allowing selection of ions (i.e., “precursor” and“fragment” ions) based on their mass to charge ratio (m/z). Quadrupole 2(Q2) is the collision cell, where ions are fragmented. The firstquadrupole of the mass spectrometer (Q1) selects for molecules with themass to charge ratios of the specific dihydroxyvitamin D metabolites tobe analyzed. Precursor ions with the correct m/z ratios of the precursorions of specific dihydroxyvitamin D metabolites are allowed to pass intothe collision chamber (Q2), while unwanted ions with any other m/zcollide with the sides of the quadrupole and are eliminated. Precursorions entering Q2 collide with neutral Argon gas molecules and fragment.This process is called Collision Activated Dissociation (CAD). Thefragment ions generated are passed into quadrupole 3 (Q3), where thefragment ions of the desired dihydroxyvitamin D metabolites are selectedwhile other ions are eliminated.

The methods of the invention may involve MS/MS performed in eitherpositive or negative ion mode. Using standard methods well known in theart, one of ordinary skill is capable of identifying one or morefragment ions of a particular precursor ion of a dihydroxyvitamin Dmetabolite that can be used for selection in quadrupole 3 (Q3).

If the precursor ion of a dihydroxyvitamin D metabolite of interestincludes an alcohol or amine group, fragment ions are commonly formedthat represent a dehydration or deamination of the precursor ion,respectfully. In the case of precursor ions that include an alcoholgroup, such fragment ions formed by dehydration are caused by a loss ofone or more water molecules from the precursor ion (i.e., where thedifference in m/z between the precursor ion and fragment ion is about 18for the loss of one water molecule, or about 36 for the loss of twowater molecules, etc.). In the case of precursor ions that include anamine group, such fragment ions formed by deamination are caused by aloss of one or more ammonia molecules (i.e. where the difference in m/zbetween the precursor ion and fragment ion is about 17 for the loss ofone ammonia molecule, or about 34 for the loss of two ammonia molecules,etc.). Likewise, precursor ions that include one or more alcohol andamine groups commonly form fragment ions that represent the loss of oneor more water molecules and/or one or more ammonia molecules (e.g.,where the difference in m/z between the precursor ion and fragment ionis about 35 for the loss of one water molecule and the loss of oneammonia molecule). Generally, the fragment ions that representdehydrations or deaminations of the precursor ion are not specificfragment ions for a particular analyte.

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theresulting mass chromatograms are similar to chromatograms generated intraditional HPLC methods. The areas under the peaks corresponding toparticular ions, or the amplitude of such peaks, are measured and thearea or amplitude is correlated to the amount of the analyte (vitamin Dmetabolite) of interest. In certain embodiments, the area under thecurves, or amplitude of the peaks, for fragment ion(s) and/or precursorions are measured to determine the amount of a dihydroxyvitamin Dmetabolite. As described above, the relative abundance of a given ioncan be converted into an absolute amount of the original analyte, i.e.,dihydroxyvitamin D metabolite, using calibration standard curves basedon peaks of one or more ions of an internal molecular standard, such as⁶D-25OHD₃.

In certain aspects of the invention, the quantity of various ions isdetermined by measuring the area under the curve or the amplitude of thepeak and a ratio of the quantities of the ions is calculated andmonitored (i.e. “daughter ion ratio monitoring”). In certain embodimentsof the method, the ratio(s) of the quantity of a precursor ion and thequantity of one or more fragment ions of a dihydroxyvitamin D metabolitecan be calculated and compared to the ratio(s) of a molecular standardof the dihydroxyvitamin D metabolite similarly measured. In embodimentswhere more than one fragment ion of a dihydroxyvitamin D metabolite ismonitored, the ratio(s) for different fragment ions may be determinedinstead of, or in addition to, the ratio of the fragment ion(s) comparedto the precursor ion. In embodiments where such ratios are monitored, ifthere is a substantial difference in an ion ratio in the sample ascompared to the molecular standard, it is likely that a molecule in thesample is interfering with the results. To the contrary, if the ionratios in the sample and the molecular standard are similar, then thereis increased confidence that there is no interference. Accordingly,monitoring such ratios in the samples and comparing the ratios to thoseof authentic molecular standards may be used to increase the accuracy ofthe method.

In particularly preferred embodiments of the invention, the presence orabsence or amount of two or more dihydroxyvitamin D metabolites in asample are detected in a single assay using the above described MS/MSmethods.

The following examples serve to illustrate the invention. These examplesare in no way intended to limit the scope of the invention.

EXAMPLES Example 1 Determination of 1α,25-dihydroxyvitamin D₃ and1α,25-dihydroxyvitamin D₂ by LC-MS/MS

50 μl of an internal standard mixture (stripped serum spiked with1α,25(OH)2D3-[6,19,19]-2H at 50 pg/50 microliters and1α,25(OH)2D2-[26,26,26,27,27,27]-2H at 200 pg/50 microliters) was addedto test tubes then 500 μl of calibrator solution, quality control testsolution, or serum standard, followed by the internal standard mixture.The solutions were delipidized by adding 50 μl MgCl₂/dextran sulfatesolution and mixing thoroughly. The tubes were then centrifuged for 20minutes and 500 μl of supernatant was transferred to ImmunoTubecartridges containing anti-dihydroxyvitamin D immunocapsules from ALPCODiagnostics (Catalog Number K2113.SI). The cartridges were incubated ona shaker at room temperature for two hours. The beads were then washedthree times with 750 μl deionized water. The beads were drained betweenwashes by centrifuging the cartridges. Dihydroxyvitamin D bound to thebeads was eluted with 250 μl ethanol directly into a glass HPLC insertand then dried to completion under nitrogen. The samples were thenderivatized by adding 50 μl of 50 microliters of4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) solution (0.8 mg/mL inacetonitrile). The derivitization reaction was stopped by adding 50 μldeionized water.

The HPLC inserts were then transferred to an HPLC autosampler forloading to the LC-MS/MS analyzer. LC-MS/MS was performed using a ThermoFinnigan LC-MS/MS analyzer (Thermo Finnigan Quantum TSQ (S/N: TQU00655))with an atmospheric pressure chemical ionization (APCI) source as thedetector. An autosampler was used to inject 90 μL of extracted samplesupernatant onto an HPLC column. Liquid chromatography was performedwith a Synergi™ Max-RP C-12 Phenomenex columns run at 0.8 mL/minute. Twomobile phase solutions were used for the HPLC: mobile phase A was 0.1%formic acid in HPLC-grade water and mobile phase B was 100%acetonitrile. The total run time was 5.00 min with the collection windowbetween 1:31-2:31 (60 seconds). The starting condition (20 seconds) was50% mobile phase A and 50% mobile phase B; the gradient (160 seconds)was from 50% mobile phase A and 50% mobile phase B to 2% mobile phase Aand 98% mobile phase B; the wash step (60 seconds) was 2% mobile phase Aand 98% mobile phase B; and the reconditioning step was 50% mobile phaseA and 50% mobile phase B.

The flow of liquid solvent exiting the HPLC column entered the heatednebulizer interface of the Thermo Finnigan LC-MS/MS analyzer and thedihydroxyvitamin D metabolites were measured using APCI in positivemode. The solvent/analyte mixture was first converted to vapor in theheated tubing of the interface. The analytes, contained in the nebulizedsolvent, were ionized (a positive charge added) by the corona dischargeneedle of the interface, which applies a large voltage to the nebulizedsolvent/analyte mixture. The ions pass through the orifice of theinstrument and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 andQ3) are mass filters, allowing selection of ions based on their mass tocharge ratio (m/z). Quadrupole 2 (Q2) is the collision cell, where ionsare fragmented.

The first quadrupole of the mass spectrometer (Q1) selected formolecules with the mass to charge ratios of 1α,25(OH)₂D₂, 1α,25(OH)₂D₃,⁶D-1α,25(OH)₂D₂ (internal standard) and -1α,25(OH)₂D₃ (internalstandard). Ions with these m/z ratios (see table below) were allowed topass into the collision chamber (Q2), while unwanted ions with any otherm/z collide with the sides of the quadrupole and are eliminated. Ionsentering Q2 collide with neutral Argon gas molecules and fragment. Thefragment ions generated are passed into quadrupole 3 (Q3), where thefragment ions of 1α,25(OH)₂D₂, 1α,25(OH)₂D₃, ⁶D-1α,25(OH)₂D₂ (internalstandard) and -1α,25(OH)₂D₃ (internal standard) were selected (see tablebelow) and other ions are eliminated. The following mass transitionswere used for detection and quantitation during validation:

TABLE 1 Mass transitions for selected dihydroxyvitamin D metabolitesAnalyte Precursor Ion Product Ion 1α,25(OH)₂D₃ 574.37 314.121α,25(OH)₂D₃- 577.37 317.12 [6,19,19′]-²H (Internal Standard)1α,25(OH)₂D₂ 586.37 314.12 1α,25(OH)₂D₂- 592.37 314.12[26,26,26,27,27,27]-²H (Internal Standard)

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theresulting mass chromatograms are similar to chromatograms generated intraditional HPLC methods.

Area ratios of the analyte and internal standards(1α,25(OH)2D3-[6,19,19′]-2H and 1α,25(OH)₂D₂-[26,26,26,27,27,27]-²H)peaks were used to construct calibration curves, which were then used tocalculate analyte concentrations. Using the calibration curves, theconcentrations of 1α,25(OH)₂D₂ and 1α,25(OH)₂D₃ were quantitated in thepatient samples.

Example 2 Intra-Assay and Inter-Assay Precision

Stock solutions of 1α,25(OH)₂D₂ and 1α,25(OH)₂D₃ were added to pooledserum to produce a Low Pool (10-15 ng/mL of each metabolite), aMedium-Low Pool (25-35 ng/mL of each metabolite), Medium-High Pool(55-65 ng/mL of each metabolite) and a High Pool (115-130 ng/mL). Fouraliquots from each of the Low, Medium-Low, Medium-High and High Poolswere analyzed in a single assay using the LC-MS/MS protocols describedin Example 1. The following precision values were determined:

TABLE 2 Intra-Assay Variation: 1α,25-Dihydroxyvitamin D₂ (1α,25(OH)₂D₂)Low Medium-Low Medium-High High 1 12 30 68 141 2 15 26 61 125 3 11 35 63110 4 11 32 67 96 Average (ng/mL) 12.4 30.6 63.7 118.1 CV (%) 16.2%11.8% 5.3% 16.5%

TABLE 3 Intra-Assay Variation: 1α,25-Dihydroxyvitamin D₃ (1α,25(OH)₂D₃)Low Medium-Low Medium-High High 1 10 30 68 125 2 14 33 59 138 3 11 35 56116 4 15 30 59 118 Average (ng/mL) 12.3 32.1 60.6 124.2 CV (%) 17.8%8.1% 8.6% 8.2%

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” “containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

1. A method for determining the amount of 1α,25 dihydroxyvitamin D₂(1α,25(OH)₂D₂) in a biological sample, when taken from a human, bytandem mass spectrometry, comprising: (a) processing a biological samplefrom a human by one or more methods to generate a processed sample,wherein at least one method used is derivatizing 1α,25(OH)₂D₂ with4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); (b) enriching derivatized1α,25(OH)₂D₂ from step (a) by high performance liquid chromatography(HPLC); (c) generating a precursor ion of the derivatized 1α,25(OH)₂D₂obtained from step (b) wherein said precursor ion has a mass/chargeratio of 586.37±0.5; (d) generating one or more fragment ions of saidprecursor ion, wherein at least one of said one or more fragment ionscomprise an ion having a mass/charge ratio of 314.12±0.5; and (e)detecting the amount of one or more of said ions generated in step (c)or (d) or both by mass spectrometry and relating the detected ions tothe amount of said 1α,25(OH)₂D₂ in said biological sample from a human.2. The method of claim 1, wherein said processing further comprisesenriching 1α,25(OH)₂D₂ by immunopurification prior to derivatization. 3.The method of claim 1, wherein the method further comprises determiningthe amount of 1α,25 dihydroxyvitamin D₃ (1α,25(OH)₂D₃) in saidbiological sample from a human by tandem mass spectrometry.
 4. Themethod of claim 3, wherein said 1α,25(OH)₂D₃ is derivatized with4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) prior to mass spectrometry;and wherein said precursor ion of said 1α,25(OH)₂D₃ has a mass/chargeratio of 574.37±0.5.
 5. The method of claim 1, wherein said biologicalsample comprises plasma or serum.
 6. The method of claim 1, wherein saidgenerating a precursor ion comprises ionizing by atmospheric pressurechemical ionization (APCI).
 7. A method for determining the amount of1α,25 dihydroxyvitamin D₃ (1α,25(OH)₂D₃) in a biological sample, whentaken from a human, by tandem mass spectrometry, comprising: (a)processing a biological sample from a human by one or more methods togenerate a processed sample, wherein at least one method used isderivatizing 1α,25(OH)₂D₃ with 4-phenyl-1,2,4-triazoline-3,5-dione(PTAD); (b) purifying the derivatized 1α,25(OH)₂D₃ from step (a) by highperformance liquid chromatography (HPLC); (c) generating a precursor ionof the derivatized 1α,25(OH)₂D₃ having a mass/charge ratio of574.37±0.5; (d) generating one or more fragment ions of said precursorion, wherein at least one of said one or more fragment ions comprise anion having a mass/charge ratio of 314.12±0.5; and (e) detecting theamount of one or more of said ions generated in step (c) or (d) or bothby mass spectrometry and relating the detected ions to the amount ofsaid 1α,25(OH)₂D₃ in said biological sample from a human.
 8. The methodof claim 7, wherein said processing further comprises enriching1α,25(OH)₂D₂ by immunopurification prior to derivatization.
 9. Themethod of claim 7, wherein the method further comprises determining theamount of 1α,25 dihydroxyvitamin D₂ (1α,25(OH)₂D₂) in said biologicalsample from a human.
 10. The method of claim 9, wherein said1α,25(OH)₂D₂ is derivatized with PTAD prior to mass spectrometry; andwherein said precursor ion of said 1α,25(OH)₂D₂ has a mass/charge ratioof 586.37±0.5.
 11. The method of claim 7, wherein said generating aprecursor ion comprises ionizing by atmospheric pressure chemicalionization (APCI).
 12. A method for determining the amount of 1α,25dihydroxyvitamin D₂ (1α,25(OH)₂D₂) in a biological sample, when takenfrom a human, by tandem mass spectrometry, comprising: (a) processing abiological sample from a human by one or more methods to generate aprocessed sample, wherein at least one method used is enriching1α,25(OH)₂D₂ by high performance liquid chromatography (HPLC); (b)generating a precursor ion of the 1α,25(OH)₂D₂ obtained from step (a)having a mass/charge ratio of 411.35±0.5; (c) generating one or morefragment ions of said precursor ion, wherein said one or more fragmentions comprise one or more ions selected from the group consisting ofions having a mass/charge ratio of 151.12±0.5 and 135.12±0.5; and (d)detecting the amount of one or more of said ions generated in step (b)or (c) or both by mass spectrometry and relating the detected ions tothe amount of said 1α,25(OH)₂D₂ in said biological sample from a human;wherein 1α,25(OH)₂D₂ is not derivatized prior to mass spectrometry. 13.The method of claim 12, wherein said processing of step (a) furthercomprises enriching 1α,25(OH)₂D₂ by immunopurification.
 14. The methodof claim 12, wherein the method further comprises determining the amountof 1α,25 dihydroxyvitamin D₃(1α,25(OH)₂D₃) in said biological samplefrom a human.
 15. The method of claim 14, wherein said 1α,25(OH)₂D₃ isnot derivatized prior to mass spectrometry; and wherein said precursorion of said 1α,25(OH)₂D₃ has a mass/charge ratio of 399.35±0.5.
 16. Themethod of claim 12, wherein said biological sample comprises plasma orserum.
 17. The method of claim 12, wherein said generating a precursorion comprises ionizing by atmospheric pressure chemical ionization(APCI).
 18. A method for determining the amount of 1α,25dihydroxyvitamin D₃(1α,25(OH)₂D₃) in a biological sample, when takenfrom a human, by tandem mass spectrometry, comprising: (a) processing abiological sample from a human by one or more methods to generate aprocessed sample, wherein at least one method used is enriching1α,25(OH)₂D₃ by high performance liquid chromatography (HPLC); (b)generating a precursor ion of the 1α,25(OH)₂D₃ obtained from step (a)having a mass/charge ratio of 399.35±0.5; (c) generating one or morefragment ions of said precursor ion, wherein said one or more fragmentions comprise one or more ions selected from the group consisting ofions having a mass/charge ratio of 151.12±0.5 and 135.12±0.5; and (d)detecting the amount of one or more of said ions generated in step (b)or (c) or both by mass spectrometry and relating the detected ions tothe amount of said 1α,25(OH)₂D₃ in said biological sample from a human;wherein said 1α,25(OH)₂D₃ is not derivatized prior to mass spectrometry.19. The method of claim 18, wherein said processing of step (a) furthercomprises enriching 1α,25(OH)₂D₃ by immunopurification.
 20. The methodof claim 18, wherein the method further comprises determining the amountof 1α,25 dihydroxyvitamin D₂(1α,25(OH)₂D₂) in said biological samplefrom a human.
 21. The method of claim 20, wherein said 1α,25(OH)₂D₂ isnot derivatized prior to mass spectrometry; and wherein said precursorion of said 1α,25(OH)₂D₂ has a mass/charge ratio of 411.35±0.5.
 22. Themethod of claim 18, wherein said biological sample comprises plasma orserum.
 23. The method of claim 18, wherein said generating a precursorion comprises ionizing by atmospheric pressure chemical ionization(APCI).
 24. The method of claim 1, wherein said processing furthercomprises enriching derivatized 1α,25(OH)₂D₂ by high turbulence liquidchromatography (HTLC).
 25. The method of claim 7, wherein saidbiological sample comprises plasma or serum.
 26. The method of claim 7,wherein said processing further comprises enriching derivatized1α,25(OH)₂D₃ by high turbulence liquid chromatography (HTLC).